Not applicable.
This invention relates to isolated nucleic acid and amino acid sequences of novel human tumor suppressors, antibodies to such tumor suppressors, methods of detecting such nucleic acids and proteins, methods of screening for modulators of tumor suppressors, and methods of diagnosing and treating tumors with such nucleic acids and proteins.
Certain tumors, benign, premalignant, and malignant, are known to have genetic components. Some of these tumors are caused by mutations or inactivation of xe2x80x9ctumor suppressorxe2x80x9d genes. In normal cells, the tumor suppressor genes are involved in the regulation of cell growth and proliferation and in the control of cellular aging, anchorage dependence and apoptosis. When the tumor suppressor genes are mutated or inactivated, cells are transformed and become immortalized or tumorigenic. These transformed cells can be reverted back to the normal phenotype (i.e., the cell growth rate is suppressed) by introducing the wildtype suppressor genes.
The first tumor suppressor gene identified was the nuclear phosphoprotein, retinoblastoma gene (Rb). Retinoblastoma is a malignant tumor of the sensory layer of the retina, and often occurs bilaterally during childhood. Retinoblastoma exhibits a familial tendency, but it can be acquired. Mutations in the Rb gene and inactivation of its product have been shown to be involved in other tumors, such as bladder, breast, small cell lung carcinomas, osteosarcomas, and soft tissue sarcomas. It was demonstrated that reconstitution of Rb-deficient tumor cells with the wildtype Rb leads to the suppression of growth rate or tumorigenicity (Huang et al., Science 242:1563-1566 (1988)). This result provides direct evidence that Rb protein is a tumor suppressor.
Another well-characterized tumor suppressor is the gene for the nuclear phosphoprotein, p53. More than half of all human cancers are associated with mutations in the tumor suppressor gene p53 (see, e.g., Hollstein et al., Science 253:49-53 (1991); Caron de Fronmentel and Soussi, Genes Chromosom. Cancer 4: 1-15; Harris and Hollstein, N. Engl. J. Med. 329:1318-1327 (1993); Greenblatt et al., Cancer Res. 54:4855-4878 (1994)). Mutations in p53 often appear to be a critical step in the pathogenesis and progression of tumors. For example, missense mutations of p53 occur in tumors of the colon, lung, breast, ovary, bladder, and several other organs. Alternatively, inactivation of the wildtype p53 proteins in cells can cause tumors. For example, certain strains of human papillomavirus (HPV) are known to interfere with the p53 protein function, because the virus produces a protein, E6, which promotes the degradation of the p53 protein.
Recently, another tumor suppressor gene, p33ING1, has been identified. p33ING1 directly cooperates with tumor suppressor gene p53 in growth regulation (Garkavtsev et al., Nature Genetics 14:415-420 (1996); Garkavtsev et al., Nature 391:295-298 (1998); U.S. Pat. No. 5,986,078, all of which are herein incorporated by reference). Neither of p53 or p33ING1 can alone cause growth inhibition when the other one is suppressed (Garkavtsev et al. (1998), supra). According to immunoprecipitation studies, p33ING1 proteins modulate the p53 activity through physical interaction. It has been also reported that some neuroblastoma cells have a mutation of the p33ING1 gene, and some breast cancer cell lines exhibit reduced expression of p33ING1 (Garkavtsev et al. (1996), supra).
Cancer remains a major public concern. Although epidemiological and cytogenetic studies demonstrated that a number of recessive genetic mutations are involved in various cancers, only a limited number of tumor suppressors have been identified. Therefore, there is a need to identify and isolate other tumor suppressor genes. The identification and isolation of new tumor suppressor genes would assist the diagnosis, prevention, and treatment of tumors and cancers.
The present invention thus provides for the first time nucleic acid and amino acid sequences of a new tumor suppressor gene called p33ING2, as well as antibodies to p33ING2, methods of detecting such nucleic acids and proteins, methods of screening for modulators of p33ING2, and methods of diagnosing and treating tumors. P33ING2 nucleic acids and proteins are tumor suppressors that play a key role in regulation of cell proliferation and tumor suppression.
In one aspect, the present invention provides an isolated nucleic acid encoding a tumor suppressor polypeptide p33ING2, wherein the polypeptide has greater than 70% amino acid sequence identity to a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In one embodiment, the nucleic acid encodes a polypeptide that selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment, the nucleic acid encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In yet another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:2. In yet another embodiment, the nucleic acid is from human. In yet another embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as degenerate primer sets encoding amino acid sequences selected from the group consisting of: SEQ ID NO:3 (MLGQQQQ) and SEQ ID NO:4 (KKDRRSR). In yet another embodiment, the nucleic acid encodes a polypeptide having a molecular weight of about 28 kDa to about 38 kDa. In yet another embodiment, the nucleic acid encodes a tumor suppressor polypeptide p33ING2 that specifically hybridizes under stringent conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2. In yet another embodiment, the nucleic acid selectively hybridizes under moderately stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2.
In another aspect, the present invention provides an isolated tumor suppressor polypeptide p33ING2, wherein the polypeptide has greater than 70 % amino acid sequence identity to a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In one embodiment, the tumor suppressor polypeptide selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide is from human. In yet another embodiment, the polypeptide is wildtype p33ING2.
In yet another aspect, the present invention provides an antibody that selectively binds to a p33ING2 polypeptide comprising an amino acid sequence of SEQ ID NO:1, but does not bind to a p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody selectively binds to a p33ING2 polypeptide comprising the amino acid sequence of SEQ ID NO:5, but does not bind to a p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8.
In yet another aspect, the present invention provides an expression vector comprising any one or more of the p33ING2 nucleic acid described herein. The invention also provides a host cell transfected with a vector comprising any one or more of the p33ING2 nucleic acid described herein.
In yet another aspect, the present invention provides a method for identifying a compound that modulates a tumor suppressor polypeptide p33ING2, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed a tumor suppressor polypeptide p33ING2, wherein the polypeptide has greater than 70 % amino acid sequence identity to a polypeptide comprising an amino acid sequence of SEQ ID NO:1; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the polypeptide. In one embodiment of the method, the polypeptide selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment of the method, functional effect is determined by measuring changes in cell growth. In yet another embodiment of the method, the polypeptide is recombinant. In yet another embodiment of the method, the polypeptide is from a human. In yet another embodiment of the method, the polypeptide comprises an amino acid sequence of SEQ ID NO:1. In yet another embodiment of the method, the cell is an HCT116 human colon cancer cell line. In yet another embodiment of the method, the cell has the missense p33ING2 sequence of a polypeptide comprising an amino acid sequence of SEQ ID NO:6.
In yet another aspect, the present invention provides a method of inhibiting cellular proliferation, the method comprising transducing a cell with an expression vector, the vector comprising a nucleic acid encoding a tumor suppressor polypeptide p33ING2, wherein the polypeptide has greater than 70 % amino acid sequence identity to a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In one embodiment of the method, the polypeptide selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment of the method, the nucleic acid encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In yet another embodiment of the method, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:2. In yet another embodiment of the method, the nucleic acid is from human. In yet another embodiment of the method, the nucleic acid encodes a polypeptide having a molecular weight of about 28 kDa to about 38 kDa. In yet another embodiment of the method, the cell has a missense or null endogenous p33ING2 phenotype. In yet another embodiment of the method, the cell has a missense p33ING2 sequence of a polypeptide comprising an amino acid sequence of SEQ ID NO:6.
In yet another aspect, the present invention provides a method of detecting the presence or absence of p33ING2 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a p33ING2-specific reagent that selectively associates with p33ING2; and (iii) detecting the level of p33ING2-specific reagent that selectively associates with the sample. In one embodiment of the method, the p33ING2-specific reagent is selected from the group consisting of a p33ING2-specific antibody, a p33ING2-specific primer, and a p33ING2-specific nucleic acid probe. In another embodiment of the method, the p33ING2-specific nucleic acid probe binds to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:7, or to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2, or to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:10. In yet another embodiment of the method, the biological sample comprises intact chromosome 4q35. In yet another embodiment of the method, the p33ING2-specific reagent detects nucleic acid, such as DNA or RNA. In yet another embodiment of the method, the nucleic acid is a polymorphic variant of p33ING2. In yet another embodiment of the method, the p33ING2-specific reagent is an antibody that selectively binds to p33ING2. In some embodiments, the antibody is polyclonal. In yet another embodiments of the method, the antibody selectively binds to a p33ING2 polypeptide comprising an amino acid sequence of SEQ ID NO:1, but not to a p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8. In yet another embodiment of the method, the antibody selectively binds to a p33ING2 polypeptide comprising an amino acid sequence of SEQ ID NO:5, but does not bind to a p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8.
In yet another aspect, the present invention provides a method of determining a test amount of p33ING2 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a p33ING2-specific reagent that selectively associates with p33ING2; and (iii) comparing the test amount to a control. In one embodiment, the control is an amount of p33ING2 in a normal cell. In another embodiment, the p33ING2-specific reagent is selected from the group consisting of p33ING2-specific antibody, a p33ING2-specific primer; and p33ING2-specific nucleic acid probe.
In yet another aspect, the present invention provides a method of detecting the presence or absence of p33ING1 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a p33ING1-specific antibody that selectively binds to p33ING1 but not to p33ING2; and (iii) detecting the level of p33ING1-specific antibody that selectively associates with the sample. In one embodiment, the p33ING1-specific antibody is polyclonal.
In yet another aspect, the present invention provides a method of determining a test amount of p33ING1 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a p33ING1-specific antibody that selectively associates with p33ING1 but not to p33ING2; and (iii) comparing the test amount to a control. In one embodiment, the control is an amount of p33ING1 in a normal cell. In another embodiment, the p33ING1-specific antibody is polyclonal.